Covalent Immobilization of Human Placental 17ß-Hydroxysteroid Dehydrogenase Type 1 onto Glutaraldehyde Activated Silica Coupled with LC-TOF/MS for Anti-Cancer Drug Screening Applications.

Human 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), a potential target in breast cancer prevention and therapy, was extracted from human placenta and immobilized on nonporous silica (∼5 µm) with a covalent method for the first time. The optimum initial enzyme concentration and immobilization time during the immobilization process were 0.42 mg mL-1 and 12 h, repectively. The binding was confirmed by scanning electron microscope (SEM) and infrared spectroscopy (FT-IR). It could improve the pH, thermal and storage stability compared to free enzyme. Moreover, the immobilized enzyme could be reused at least four times. A screening method based on it coupled with liquid chromatography-time-of-flight mass spectrometer (LC-TOF/MS) was established, and the half-maximal inhibitory concentration (IC 50) of apigenin for the immobilized enzyme was 291 nM. Subsequently, 10 natural products were evaluated leading to inhibition of the activity of 17ß-HSD1 at the concentration of 25 µM, and six of them inhibit the activity over 50%.